Cytochrome P-450 is the terminal oxidase in the liver microsomal monooxygenase system. It is a key protein in the metabolism of xenobiotics, steroids, and fatty acids, in the metabolism and possible activation of chemical carcinogens and in the changes in the rate of metabolism of these substances induced by various xenobiotics. The overall objective of this research is to understand the regulated biosynthesis of cytochrome P-450 and the structural relationships between the different species of cytochrome P-450. In this initial proposal we plan to study the structure and function of the genes for the two major forms of cytochrome P-450 that are inducible by phenobarbital and polycylic hydrocarbons in rabbit liver. The mRNAs for the cytochromes P-450 will be partially purified from livers of rabbits pre-treated with phenobarbital or beta-naphthoflavone by sucrose gradient centrifugation and oligo (dT) cellulose affinity chromatography. Double-stranded cytochrome P-450 cDNA will be produced by sequential incubation of the mRNA template with reverse transcriptase and E. coli DNA polymerase. Cytochrome P-450 cDNA will be isolated and amplified by molecular cloning with appropriate transformed clones being selected by hybrid-arrest translation techniques. The structure of the cDNA will be analyzed by restriction enzyme mapping and sequence analysis. Fragments of rabbit genomic DNA containing cytochrome P-450 gene sequences will then be isolated by molecular cloning and the restriction maps of genomic DNA will be compared with that of the cDNA genes. The cloned cDNA will also be used to analyze the concentrations of cytochrome P-450 mRNA in rabbit liver after administration of phenobarbital and Beta-naphthoflavone to gain some insight into the mechanism involved in the induction of cytochrome P-450 activity by these agents.